Growth hormone (GH) is a hormone that stimulates growth and cell reproduction in humans and other animals. It is a 191-amino acid, single chain polypeptide hormone which is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. The hormone is also known as somatotropin when referring to growth hormone produced by recombinant DNA technology, and is abbreviated “rhGH”.
Growth hormone has a variety of functions in the body, the most noticeable of which is the increase of height throughout childhood, and there are several diseases which can be treated through the therapeutic use of GH.
The effects of growth hormone deficiency vary depending on the age at which they occur. In children, growth failure and short stature are the major manifestations of GH deficiency. It can also cause sexual immaturity. In adults the effects of deficiency are more subtle, and may include deficiencies of strength, energy, and bone mass, as well as increased cardiovascular risk.
There are many causes of GH deficiency, including mutations of specific genes, congenital malformations involving the hypothalamus and/or pituitary gland, and damage to the pituitary from injury, surgery or disease.
Deficiency is treated through supplementation with external GH. All GH in current use is a biosynthetic version of human GH, manufactured by recombinant DNA technology. GH is used as replacement therapy in children and adults with GH deficiency of either childhood-onset (after completing growth phase) or adult-onset (usually as a result of an acquired pituitary tumor). In these patients, benefits have variably included reduced fat mass, increased lean mass, increased bone density, improved lipid profile, reduced cardiovascular risk factors, and improved psychosocial well-being.
Genentech Inc (US) was the first to clone rhGH and this was described in patent EP-B 22242. As of 2006, synthetic growth hormones available in the United States and Europe (and their manufacturers) included Nutropin (Genentech), Humatrope (Eli Lilly), Genotropin (Pfizer), Norditropin (Novo Nordisk), Saizen (Merck Serono), and Omnitrope (Sandoz).
Although molecular biological techniques have dramatically increased the availability of many proteins and/or polypeptides (hereinafter referred to as proteins), the therapeutic use of said proteins is often times hindered by other factors, such as short plasma half-life due to renal and receptor-mediated clearance, aggregation, proteolytic degradation, poor bioavailability and physical properties which preclude efficient formulations.
A mechanism for enhancing protein availability is by conjugation of the protein with derivatizing compounds, which include, but are not limited to, polyethylene glycol and polypropylene glycol. Some of these benefits recognized include: lowered immunogenicity and antigenicity, increased duration of action, and altered pharmacokinetic properties. [Veronese, F. M. “Enzymes for Human Therapy: Surface Structure Modifications,” Chimica Oggi, 7:53-56 (1989)] (Herein reference 5).
By PEGylating rhGH, it may be possible to improve the characteristics of the molecule for medical use by increasing its in vivo half-life (hereby achieving reduced dosage or reduced frequency of dosing), improving its stability and decreasing its antigenicity or a combination thereof.
Generally, this type of modification to a molecule is well known in the art and there are numerous patents available in the patent literature, describing this concept. For example a PEGylated Erythropoietin (EPO) from Hofmann La Roche is described in EP-B 1196443 claiming a specific linker comprising PEG covalently bound to EPO, a PEGylated interferon alpha described in EP-B 975369 from the company Nektar/La Roche and another PEGylated interferon alpha in EP-B 1562634 from the company Hofmann La Roche.
In vivo clearance of rhGH is believed to occur by the following two mechanisms. The first is renal clearance where rhGH is cleared from the circulation by renal glomerular filtration. Renal clearance of rhGH is well documented and PEGylation of synthetic rhGH is therefore an obvious choice to solve this problem. Renal clearance accounts for around 25-53% of the total clearance of rhGH (Girard, J. Mehls, O. J. Clin. Invest. 1994 March; 93(3): 1163-1171, reference 3 herein.)
The second mechanism is hepatic clearance (liver). Hepatic GH uptake occurs by receptor-mediated endocytosis followed by lysosomal degradation.
A third mechanism is receptor mediated clearance in other tissue such as chondrocytes of the cartilage. By reducing the binding affinity of GH to the GH receptor by PEGylation, the receptor mediated clearance will be reduced.
However, there are dedicated problems with the administration of rhGH. One major disadvantage of subcutaneously administrated rhGH is the occurrence of lipoatrophy in patients receiving the treatment.
Lipoatrophy is the medical term used for localized loss of fat tissue. Subcutaneously administered rhGH formulations have displayed lipoatrophy problems, which is believed to be caused by high local concentration of the growth hormone complex and at the injection site.
Büyükgebiz A. et al published in J. Pediatr. Endocrinol. Metab. 1999 January-February; 12(1):95-7 describes such a medical record (herein reference 1). This is a report of a patient with isolated GH deficiency due to 6.7 kb gene deletion who received high dose rhGH treatment and developed local lipoatrophies at injection sites without any antibody detection after 6 years of therapy. The etiology of the lipoatrophy is suspected to be by the direct lipolytic effect of high doses of rhGH.
Lipoatrophy related to the administration of rhGH is believed to be caused by the rhGH activity itself, by higher concentrations and by prolonged exposure. These higher concentrations occur near injections sites.
The chance that high growth hormone activity accumulates near the injection site is even higher in case that rhGH is PEGylated because of an increased residence time. In the case of PEGylated rhGH formulations, the tissue will experience a sustained and increased exposure to growth hormone activity, due to the fact that the PEGylated conjugate possess activity necessary for pharmacological activity and the conjugate is diffusion limited due to the conjugate size. The outcome is increased lipolysis at the injection site.
WO-A 2005/079838 describes pegylated hGH, wherein the hGH moiety is attached to a polyethylene glycol polymer via amino functional group, which can be considered as permanent attachment due to the stability of the amino group. An example of such a PEGylated hGH compound, which exhibits lipoatrophy, is the compound PHA-794428. Compound PHA-794428 is a PEGylated rhGH and also described in WO-A 2005/079838 from the company Pharmacia (acquired by Pfizer) and further described in Horm. Res. 2006; 65 (suppl. 4): 1-213, CF1-98 GH/IGF Treatment with title “First in-human study of PEGylated recombinant human growth hormone”, Philip Harris et al. (herein reference 4).
According to the clinical trial information as published on www.clinicaltrials.gov, the trial was terminated on 10 Dec. 2007. Pfizer's decision to terminate the program was due to cases of injection-site lipoatrophy that were reported in the clinical Phase 2 studies after a single injection of PHA 794428.
WO-A 2006/102659 (Nektar) also describes and suggests rhGH-PEG conjugates (linear and branched types) via amide bond. The problem to be solved in WO-A 2006/102659 is described in paragraph [0005] on page 2. According to the applicant, the problem to be solved is reduced dosing frequency. Since rhGH therapy typically requires daily injections, patients, and in particular, pediatric patients, dislike the inconvenience and discomfort associated with this regimen. The solution described in Nektar's WO-A is the provision of new PEG-rhGH conjugates.
In table 6, [0257] of the WO-A it can be seen that the PEG-rhGH conjugates have a relatively low activity in vitro as compared to the native growth hormone without PEG. Despite the low in vitro activities, the PEGylated rhGH conjugates were active in vivo. In relation to this reads section [0261]: “Although the preliminary in vitro results suggest that increasing the amount of PEG attached to hGH reduces its ability to stimulate the hGH receptor, based on the preliminary in vivo results, it appears that a reduction in bioactivity is more than balanced by increased half-life and/or plasma availability, thus leading to a conclusion that the conjugates provided herein possess a superior pharmacodynamic effect in vivo when compared to unmodified rhGH at an identical dosing regimen”.
WO-A 2006/102659 (Nektar) does not describe auto-cleavable linkers—i.e. it is simply observed that PEG-rhGH conjugates are active in vivo although their in vitro activities are significantly reduced. The problem of lipoatrophy is not addressed.
A solution to the challenge of engineering the desired properties of reduced lipoatrophy and reduced injection frequency into a PEGylated conjugate of hGH is the use of a prodrug approach. A prodrug is any compound that undergoes biotransformation before exhibiting its pharmacological effects. Prodrugs can thus be viewed as drugs containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule. In this case, a polymeric carrier would transiently reduce the activity of growth hormone and consequently reduce the likelihood of tissue lipolysis. Transient conjugation to a polymeric carrier would at the same time extend the half-life of the conjugate and therefore provide for a long-acting delivery of hGH.
Numerous macromolecular prodrugs are described in the literature where the macromolecular carrier is linked via a labile ester group to the medicinal agent (e.g. Y. Luo, M R Ziebell, G D Prestwich, “A Hyaluronic Acid—Taxol Antitumor Bioconjugate Targeted to Cancer Cells”, Biomacromolecules 2000, 1, 208-218, J Cheng et al, Synthesis of Linear, beta-Cyclodextrin Based Polymers and Their Camptothecin Conjugates, Bioconjugate Chem. 2003, 14, 1007-1017, R. Bhatt et al, Synthesis and in Vivo Antitumor Activity of Poly(L-glutamic acid) Conjugates of 20(S)-Camptothecin, J. Med. Chem. 2003, 46, 190-193; R. B. Greenwald, A. Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A. Martinez, K. Shum, S. Guan, J. Med. Chem., 1999, 42, 3657-3667; B. Testa, J. M: Mayer in Hydrolysis in Drug and Prodrug Metabolism, Wiley-VCH, 2003, Chapter 8) In theses cases, the conjugated functional group of the bioactive entity is a hydroxyl group or a carboxylic acid.
Especially for biomacromolecules but also for small molecule polymer prodrugs, it may be desirable to link the macromolecular carrier to amino groups (i.e. N-terminus or lysine amino groups of proteins) of the bioactive entity. This will be the case if masking the drug's bioactivity requires conjugation of a certain amino group of the bioactive entity, for instance an amino group located in an active center or a region or epitope involved in receptor binding. Also, during preparation of the prodrug, amino groups may be more chemoselectively addressed and serve as a better handle for conjugating carrier and drug because of their greater nucleophilicity as compared to hydroxylic or phenolic groups. This is particularly true for proteins which may contain a great variety of different reactive functionalities. In this case non-selective conjugation reactions lead to undesired product mixtures which require extensive characterization or purification and may decrease reaction yield and therapeutic efficiency of the product.
Prodrug activation may occur by enzymatic or non-enzymatic cleavage of the labile bridge between the carrier and the drug molecule, or a sequential combination of both, i.e. an enzymatic step followed by a nonenzymatic rearrangement.
In WO-A 2005/099768 PEGylated linker molecules with auto-cleavable linkers for a large group of biomolecules including somatropins (claim 6) are described. In WO-A 2005/099768, the problem to be solved is the interpatient variability and unpredictable effect of prodrug activation when enzymatic mechanism is involved (page 12, line 17-30). This application describes as a solution an aromatic linker, which may be PEG based. This linker-PEG binds the drug in a way that the drug activity is significantly reduced. It is activated only on release of the drug, which is initiated by hydrolysis. The hydrolysis rate can be controlled chemically. No special emphasis is given on GH and relevant problems, like lipoatrophy, in relation to this as such.
In summary, none of the above mentioned citations describes a solution to develop a long-acting rhGH, based on a prodrug conjugate that can be administered less frequently without increasing the frequency of lipoatrophy.
Thus an object of the present invention is the provision of such a prodrug or a pharmaceutical composition comprising said prodrug to reduce the administration frequency of rhGH using PEG conjugated to rhGH without significantly inducing lipoatrophy.